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Book. 



SPOROZOON PARASITES OF CERTAIN FISHES IN 
THE VICINITY OF WOODS HOLE, MASSACHUSETTS 

By C. W. Hahn 

From . BULLETIN OF THE BUREAU OF FISHERIES, Volume XXXIII, 1913 
Document No. Sio : : : : : : : Issued April 29, 191s 




WASHINGTON ;:::;: GOVERNMENT PRINTING OFFICE 



1915 



SPOROZOON PARASITES OF CERTAIN FISHES IN 
THE VICINITY OF WOODS HOLE. MASSACHUSETTS 

By C. W. Hahn 

From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXXIII, 1913 
Document No. 810 : : : : ; ; ; ; ; ; ; ; ; ; : Issued April 2g, 191 5 




WASHINGTON 



GOVERNMENT PRINTING OFFICE 



1915 



W»n»gr»^ 



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SPOROZOON PARASITES OF CERTAIN FISHES IN THE VICINITY 
OF WOODS HOLE, MASSACHUSETTS 

By C. W. Hahn 



191 



CONTENTS. 

Page. 

Occurrence of disease 193 

Methods of study 194 

Experiments to determine character of infection 195 

Pathological condition of the tissues 197 

Bacteria associated with atrophied tissues 199 

Sporozoa associated with atrophied tissues 201 

Stages of Myxobolus musculi 202 

ChloromjTcum funduli 205 

Protozoa related to those here described 206 

Prevalence of m)rxosporidian infection in fish 208 

General conclusions 209 

Bibliography 210 

Explanation of plates 212 

192 



SPOROZOON PARASITES OF CERTAIN FISHES IN THE VICINITY 
OF WOODS HOLE. MASSACHUSETTS. 

J- 

By C. W. HAHN. 
J- 

While studying the Sporozoa in different species of fish at Woods Hole, Mass., in 
1909, the myxospore of one was observed in diseased killifish, Fundulus heteroditus 
and Fundulus majalis. Additional material was obtained and some special experiments 
were carried out during the seasons of 1910, 191 1, 1912, and 1913, the United States 
Bureau of Fisheries providing the facilities for this and other similar studies at its 
Woods Hole biological laboratory. " 

OCCURRENCE OF DISEASE. 

When a number of Fundulus of either of the common species {heteroditus or 
majalis) are confined in aquaria for a few days during the warm season, one or more 
thickened white or pink areas appear upon the integument of some of the fishes. The 
scales of these patches are more or less loosened. They increase in size and number, and 
the number of afflicted fishes also increases. The fins when involved become bloody and 
the fin-rays are exposed. Elsewhere the integument disintegrates and the flesh is laid 
bare. Considerable excavations into the body muscle are not uncommon. The largest 
cavity of this kind obser\'ed was in the head region, measuring about 10 to 12 mm. in 
diameter and 2 to 3 mm. in depth. Such excavations expose large areas of the skull. 
When other parts are attacked, loss of blood or penetration of vital parts causes death 
before the lesion becomes conspicuous externally. The integument is thickened around 
the sores where the scales are loose. Its color is pink or white. The scales fall out at 
the edge of the sores. The caudal fin may be completely removed, also the flesh and 
integument of the tail, thus exposing the vertebrae, before the fish succumbs to the 
disease. Fish frequently give evidence of weakness and depression even before the 
flesh has been exposed. There is nothing peculiar about the locomotion except a dimin- 
ished activity. In certain cases where there is conspicuous inflammation of the integ- 
ument, especially under the head, the fish may be obser\^ed to dart downward, and, 
with a slight rotation or twist of the body, to scrape the ventral or lateral portion of 
the head upon the bottom of the aquarium. The fish slowly lose strength, the smaller 
ones first, and the larger ones not until they are greatly mutilated. Apparently all 
afiiicted fish die unless special care is given to cleanliness, water, and food. 

The proportion of fish that are diseased when caught has not been ascertained. 
The ratio of those that develop integumentary- sores in the first day or two to those 
that are healthy depends to a great measure upon the injuries received in handling the 

<^ Valuable assistance from Dr. Edward Liatoa and Mr. Vinal E. Edwards is gratefully acknowledged. 



194 BULLETIN OF THE BUREAU OF FISHERIES. 

catch. Sometimes 50 per cent of the Funduhis that have been roughly handled, as 
when stripped for eggs, will become diseased in 24 hours. Of these, half may be dead 
within 12 hours. If a few crabs happen to be confined in the same aquarium with a 
large number of Fundidus, they inflict injuries upon practically all the fish and all are 
soon diseased. Uninjured Ftmdulus develop the disease infrequently. (See p. 196.) 
Roughly speaking, 3 to 4 per cent of the Futidulus that are brought into the laboratory 
at this season (July and August) and confined in small aquaria having but a liter or 
two of water for each fish, will be found diseased within two days. Within another 
day or two some of these fish die and a large number die in the course of a week. Dis- 
eased Fundidus are therefore almost constantly available. 

METHODS OF STUDY. 

Both fresh and preser\'^ed tissues were examined microscopically, the method of 
handling the tissues being as follows: The scales having been removed with forceps, 
the edge of a slide is drawn over a diseased area with a little pressure, and the mucus 
and cellular material thus obtained is spread evenly over the surface of another slide; 
or, a portion of integument or muscle which has been removed with a scalpel is ground 
between two thick slides by giving to the upper slide a circular motion. It is necessary 
to use considerable pressure, and at times cut tough fragments with the sharp edge of 
the upper slide. Under these conditions, both slides may be preserved for observation 
and still others made from the ground-up material. Some of the smear preparations 
made in this manner were examined while fresh and others were fixed and stained. 
Altogether about 85 fish were examined microscopically. Fresh smears which were 
sometimes supplied with bile and serum were sealed with vaseline, and could then be 
examined from time to time, during a period of 24 hours. 

For sectioning, tissues were fixed in a saturated solution of corrosive sublimate in 
35 per cent alcohol with 0.2 per cent acetic acid and 6 per cent formaldehyde; also in 
the ether-formalin-alcohol mixture given below. Some of the smears were fixed in the 
same sublimate mixture; others in a solution of corrosive sublimate in 2 parts abso- 
lute alcohol and i of ether; still others in a mixture of absolute alcohol (60 per cent), 
ether (35 per cent), and strong formaldehyde (5 per cent). The mercury preparations 
are stained with a modification of Mayer's haematein. (See Hahn, Archiv fiir Protis- 
tenkunde, bd. xvii, no. 3, p. 316, footnote.) Usually the alcohol and formalin prepa- 
rations are stained in methylene blue or Giemsa's stain. The methylene blue was 
extracted in a saturate alcoholic (70 per cent) solution of both eosin and orange G. 
The Giemsa was washed in water, allowed to dry, and decolorized in carbol-xylol, 
without the use of alcohol. Some of the more recent preparations fixed by either of 
the above fluids have been more successfully stained by first treating with haematein 
for several hours, then decolorizing in 70 per cent alcohol with i per cent HCl, returning 
through the alcohols to water, and staining in methylene blue or toluidin blue. After 
dehydration they were left in a contrast stain (eosin and orange G) for a few minutes 
and rapidly run into 95 per cent alcohol, carbol-xylol, and two changes or xylol. Both 
smear preparations and sections are mounted in Canada balsam without cover glasses. 

Searching is most satisfactorily carried on with an ocular of i inch and an objective 
of one-fifth inch focal distance. A one-twelfth inch oil immersion objective combined 
with the same ocular for ordinary observation is supplemented, when occasion requires, 
with a no. 2 compensating ocular. The one-fifth inch objective is not too high to be 



SPOROZOON PARASITES OF FISHES. 1 95 

used without a cover glass and reveals most of the details necessary to recognize the 
presence of Protozoa or other unusual histological conditions. A mechanical stage is 
indispensable. 

Three organisms are involved in most of the Fundidus ulcers, rarely a fourth. A 
thick, short bacillus is the most abundant. A long, slender bacillus is less common. 
The Sporozoa are represented by a species of Myxoholus, and in one case a species of 
Chloromyxum. From the evidence in the following account it will be learned that the 
primary attack upon healthy tissues, in a certain proportion of the diseased fish, is prob- 
ably made by the long bacillus. At least a few and probably many of the diseased fish 
are primarily attacked by Mjrxosporidia. The short bacillus is more or less incapable 
of rapid growth in living cells of any kind. While it is not within our province to make 
an exhaustive study of the fungus diseases, it has been necessary to ascertain to what 
extent they participate in bringing about these pathological conditions. 

EXPERIMENTS TO DETERMINE CHARACTER OF INFECTION. 

The following experiments were carried out in order to gain some accurate infor- 
mation as to the conditions whereby healthy fish are infected and the possibiUties of 
their recovery. At the time it was not possible to discriminate between fish that were 
infected by a fungus and those that were infected by a sporozoon. It will be apparent 
that the experiments are not vitally affected by the kind of parasite present. 

Forty fish were divided equally and placed in two 5-gallon aquaria. These fish 
had been seined in the usual manner and brought to the laboratory on board the steamer 
Phalarope in large milk cans. The trip from the collecting grounds (Menemsha Bight) 
usually requires about one and one-half hours. The cans accommodate from 200 to 
300 fish each. A hose supplies them with fresh water. The 40 fish used in this case 
were examined carefully and found to be free from all visible integumentary disturbance. 

First stage. — Aquarium no. i was carefully cleaned and sterilized. Aquarium no. 2 
had contained diseased fish, and 2 diseased fish were allowed to remain with the 20 
fish used in the experiment. Contaminated fish from other sources were always kept 
in this jar. Both groups were fed about every 48 hours. After a period of 1 1 days 
none of the fish in the clean jar showed any signs of disease. From this fact we con- 
cluded that they were free from the disease and suitable for experimentation of a dif- 
ferent kind. After the same period (11 days) the contaminated jar had one fish with a 
conspicuous sore. It died a day later. 

Second stage. — On the eleventh day one of the fish in each of the two jars was 
operated upon. A scale or two was removed and the integument pierced with a scalpel 
just back of and dorsal to the opercle. More diseased fish were introduced into aquarium 
no. 2. Five days later the fish in aquarium no. i which had been operated upon died. 
The integument, at the point where the incision had been made, had developed a typical 
sore. At this time the fish with the pierced integument in no. 2, being a large fish, had 
not developed a sore of noticeable extent. 

Third stage. — On the sixteenth day of the experiment, all fish having recovered in 
both no. I and no. 2, scales were removed and the integument of all the fish was pierced 
in the same manner as was done with the two above mentioned. Two days later almost 
all of those in jar no. 2 had developed marked diseased patches at the very spot where 
the integument had been pierced. No noticeable change had taken place in the fish of 
the clean jar. Four days later one fish in jar no. i died from the eflEects of the rapidly 

64023°— 15 — 2 



196 BULLETIN OF THE BUREAU OF FISHERIES. 

advancing disease. Subsequent examinations of the tissues showed that the probable 
cause of this disease is a myxosporidian belonging to the genus CUoromyxum, being 
unique in this respect. (See p. 205.) Four dead fish taken from jar no. 2 at this time 
included two that had been introduced for the purpose of spreading the disease. After 
seven days the fish in jar no. i were all recovering. The incised integument had closed 
and appeared a little white. Of those in jar no. 2, two were dead, three were seriously 
diseased and died within 24 hours, and the others had conspicuous sores. The remaining 
14 fish from this time began to show signs of recovery, probably because they were not 
subjected to contamination and they were fed more regularly. Twelve fish remained 
in jar no. i and had completely recovered before the experiment was discontinued. 

In the above experiment the treatment given to the two jars was as far as possible 
the same. Some fish escaped from both jars by jumping out. 

The first stage of this experiment, which corresponds with the first 1 1 days, was not 
conclusive. One fish, having contracted a fatal disease from a contaminated en^'iron- 
ment, demonstrates the possibility that fish with apparently healthy integument may 
acquire the ulcers. The second stage of the test, covering six days, was still less con- 
clusive. But the third stage, covering seven days, showed beyond doubt that the infec- 
tion enters a lesion of the integument, that contamination favors its entrance, that 
some of these diseases may be contracted in tolerably pure water, and that lesions 
which are not contaminated heal completely. 

Another experiment of this character was then started, making use of some of all 
the lots of fish that had been under observation. All were in good condition. Eight 
fish of fair size were carefully removed from this stock and, by means of a small steril- 
ized scalpel, an incision was made back of the head and a pocket then made under the 
integument so as to disturb the tissues as little as possible. Into this pocket was inserted 
a bit of the diseased flesh from sores of four fish taken from different aquaria. As a 
control, eight more fish of the same size were similarly cut, but nothing was introduced 
into the pockets. Of the contaminated fish, four died from the disease in two days, 
the balance in four days. In this case the disease spread over the whole upper part of 
the body and assumed the characteristic appearance usually encountered. Only one of 
the controls died. The others healed and recovered completely. From time to time the 
diseased fish which were introduced into the contaminated jar and those used for the 
inoculation experiments were examined microscopically. All were infected with bacteria. 

This last experiment, covering a period of four days, confirms the results of the 
previous experiments as to the infectious nature of the disease as well as the inability 
of the fish to throw off strong cultures of the causal agents. We also learn that when 
the fish is well nourished and in a wholesome environment, it has considerable natural 
immunity and recovers readily from the affliction. 

In order to prevent the customary mortality from this kind of affliction, care 
should be taken not to injure the fish while collecting; no crabs or other carnivorous 
enemies should be confined in the same tanks with the Futidtdus, and after establishing 
them in an aquarium without crowding, they should be fed on alternate days. The 
aquarium should be kept free from dead and diseased fish. With proper circulation of 
■water, this treatment will no doubt reduce the mortality to a negligible quantity and 
preserve the fish for several months. 



SPOROZOON PARASITES OF FISHES. 1 97 

PATHOLOGICAL CONDITION OF THE TISSUES. 

Those typical sores in which Sporozoa can not be positively demonstrated, and of 
which a part may be due to bacteria, present the following histological conditions. They 
are probably primarily exogenous ulcers in which there is at times abundant granular 
degeneration derived both from lymphocytes and haemocytes. Sometimes at the nidus 
of the necrotic area there are small cysts or abcesses containing small lymphocytes. 
Usually the vascular tissues abound and erythrocytes preponderate. There is a decided 
tendency at times for the epidermis to form a cicatrix. Again it gives evidence of 
sloughing off. But so far as the muscle tissue is concerned universal necrosis is common. 

The involved epidermis contains numerous nonstaining globules or masses of 
variable size (fig. 36, pi. xxi), as to the exact nature of which we are yet in doubt. They 
are also to be found in the connective tissue of the dermis and in certain partly atrophied 
muscle fibers when adjacent to degenerate tissue. The}' seem to be more numerous in 
the epidermal cells wherein there are obvious signs of disintegration (pp. 198, 201, 203). 
Inasmuch as there is a nonstaining zoogloea or secretion about some of the bacilli that are 
commonly found in these parts, which frequently prevents them from staining (see p. 
200), it is possible that these bodies are of the same nature and contain one or more of the 
bacilli. No doubt many are fat globules, but some are certainly not. Some of these 
bodies in sections of muscle containing myxoplasms possess a well-defined nucleus. 
(Fig. 12, pi. XX.) 

In smears of integument, it is occasionally possible to find fragments of considerable 
size having the epidermal cells more or less filled with the short bacillus referred to above. 
It is not difficult to prove, by the observation of fresh material or by comparison of tis- 
sues of different stages of degeneration, that the short bacillus is seldom found in normal 
living cells. It is therefore not probable that the primary attack upon the epidermis is 
caused by this particular organism. The long slender bacillus is less commonly en- 
countered in the dermis and epidermis. There is but little evidence in support of the 
view that it is the initial cause of epidermal decadence. 

The muscle fibers beneath these infected areas present an interesting condition. To 
the naked eye there seem to be numerous white threads running parallel with the muscle 
cells. This is especially true of well-advanced ulcers. When seen under the microscope, 
such flesh has but few normal fibers with fibrillae and cross striae. Most of them have the 
sarcolemma and interfibrillar connective tissue still sufficiently intact to retain the general 
external structure of the separate fibers, but the myoplasm is in various stages of de- 
generation. We conclude, therefore, that the parasite is intracellular and does not pass 
readily from one fiber to another. The muscle fibers sometimes undergo degeneration 
more or less uniformly throughout their length. In some cases it is more rapid in the 
immediate ^dcinity of the parasites. This we know from sections where the fibrillae show 
in places adjacent to degenerate myoplasm in which Sporozoa are numerous. One side 
or the middle may be far more degenerate than the rest of the fiber. The parasites have 
probably passed through these regions. The first indication of change is the loss of 
fibrillation. It is rather difficult to find a parasitized fiber showing normal fibrillation 
(fig. 13, pi. XX). The pale bands of muscle fibers next become granular (figs, i and 2, 
pi. xx) and at length the sarcous elements break up into large pieces. Eventually there 
is total granular atrophy of the fiber within the sarcolemma. In certain cases, usually 



198 BULLETIN OF THE BUREAU OF FISHERIES. 

when the atrophy is hyalin, there are considerable clefts in the sarcoplasm. (Fig. 4, 
pi. XX). These spaces may come to be more or less closely packed with erythrocytes or 
leucocytes, or both, so that when the cytoplasm of the blood cells has degenerated a 
third and common condition is encountered. The nuclei in various stages of degenera- 
tion become densely packed and enlarged. They assume amoeboid shapes, large alveoli 
appear in them, and eventually they fall a prey to the short bacillus (fig. 5 and 6, pi. xx) 
elsewhere encountered. 

The conditions thus presented are such as to suggest an amoeboid parasite which 
has demolished a muscle fiber and simultaneously broken up into innumerable bacillus- 
shaped spores by schizogony. (See fig. 10, pi. xx.) The connective tissue nuclei of the 
flesh and integument and the nuclei of the gill epithelium give rise to the same degen- 
eration phenomena. Such nuclei may be about equally hypertrophied and massed in 
such a manner as to completely disguise their true nature. Both muscle and vascular 
nuclei may occur in abnormal numbers under the sarcolemma of fibers which are in 
almost any state of atrophy but without clefts. (Fig. 5, pi. xx.) 

In both fresh and stained muscle the evolution of a curious artifact was observed. 
It appears as a dense hyalin body in the sarcolymph, between fibrillae. (Fig. 3, pi. xx.) 
Assuming an amoeboid form it resembles a rapidly growing organism. (Fig. 2 and 7, 
pi. XX.) But the regular distribution (fig. 2 and 3) and numerous variations toward a 
crystalline rosette structure are conclusive evidence of their lifeless nature. 

Whatever the active cause of the degeneration of muscle fiber, be it bacteria or 
Protozoa, the atrophy advances far into one or more muscle fibers without causing any 
damage to the adjacent fibers. In cross sections of such tissues there may be a small 
group of normal fibers cut in section amongst numerous others that are wholly degenerate. 
Capillaries, arteries, veins, and sheets of connective tissue, entirely normal in appear- 
ance, may also penetrate these necrotic masses. This is no doubt due to the restraining 
influence of the sarcolemma upon either the parasite or toxin. As we have already 
noted, the sarcolemma retains its normal relations in completely atrophied fibers. 

Restricting our statements to tissues known to be infected by Sporozoa, there are 
but two kinds where their action has been observed, namely, muscle, and the connective 
tissue of the gill. The pathological condition of the muscle tissue, in such cases, is 
not distinguishable, as far as we know, from that which results from the action of 
bacteria; but if the pathological changes are to be considered as characteristic of a 
parasite when it is known to be the cause of the atrophy, a careful study of those cases 
where bacteria are a negligible factor is important. The myxospores, which are the 
most easily recognized stages of the Myxosporidia, are common only in smear prepara- 
tions and only those which include more or less diseased muscle fibers. These same 
smear preparations also contain cells identical in appearance to myxoplasms, pansporo- 
blasts, and sporoblasts, which happen to be the only representatives of the Sporozoa that 
we have encountered in sectioned material, thus suggesting their myxosporidian character. 

Several fragments of tissue, the integument of which was slightly diseased, were 
sectioned. They give no evidence of myxospores, but the muscle fibers present prac- 
tically the same degenerative changes to be seen elsewhere. The dermis contains 
numerous minute unstained lens-shaped structures similar to those described on 
page 197. These extend into the ends of the adjacent muscle fibers, becoming less 
numerous in the deeper parts. Such fibers show obvious signs of atrophy. Elsewhere 



SPOROZOON PARASITES OF FISHES. 1 99 

there are numerous deep fibers containing many large cells, which vary in size and have 
conspicuous nuclei. (Fig. i8, pi. xxi.) These are confined by the sarcolemma to a 
very few fibers and extend for a long distance through them. A small cavity only is 
excavated about each cell. They are usually isolated, though two or more may occupy 
the same cavity. The sarcoplasm in such cases is much atrophied, being uniformly 
granular or homogeneous. A sharp line of demarcation exists between the infected 
and uninfected parts of the muscle fiber, the former being degenerate and the latter 
striated and normal. Situated amongst the fibers containing the Protozoa are others 
lacking them but atrophied in a typical manner, the sarcoplasm being broken into 
irregular fragments. There are several other foreign and unnatural structures in the 
sections just referred to, about which the details are given on page 203. Muscle fibers 
packed with blood tissues and degenerate nuclei have not been found in any of the 
sectioned tissues which contain unmistakable cases of Myxosporidia; but no special 
significance has been attributed to this fact. 

Smears of gill filaments stained with Giemsa stain present the following conditions: 
Both normal and degenerate tissues are encountered. In some places the cartilage 
supporting structures have been attacked and are partly disintegrated. The general 
external form of the supporting tissue, including the surrounding connective tissue and 
epithelium, are, as a rule, partly maintained; but elsewhere the degeneration is com- 
plete. Epithelium and connective tissue cells disappear completely, leaving the elastic 
fibers and blood elements. Here, as elsewhere, the nuclei of the latter are most persistent, 
especially those of the erythrocytes. A large portion of the expressed fluids is composed 
of an acidophile substance containing odd-shaped portions of the fused nuclei. The 
spaces between the chromatin threads of the latter having become much dilated, fuse 
and form large masses of network. These are mechanically separated on crushing the 
tissue. Such masses of nucleic acid or degenerate chromatin have unbroken connections 
with the normal blood in the arteries or veins of the less disturbed tissue. Where the 
blood emerges from partly degenerated blood vessels, they are filled with atrophied 
erythrocyte nuclei. It seems probable that very large masses of homogeneous eosinophil 
material, which are constantly associated with the degenerate gill tissue, are derived 
from haemoglobin, lecithin, etc., of the stroma. 

Myxospores abound in these degenerate gill tissues, especially in the purulent 
residues of degeneration where nothing else remains recognizable. They also occur 
deep in the connective tissue near the cartilage and amongst the capillaries. The 
spores, developing spores, sporoblasts, and pansporoblasts, in all stages, are clearly 
defined, apparently unaffected by the conditions where tissue cells have become wholly 
atrophied. This fact, together with the great abundance of m3rxosiX)res and developing 
myxospores, both occurring in considerable clusters, prove beyond question that the 
primary cause of necrosis in this case is the Myxobolus. No bacteria or other possible 
agents have been encountered. 

BACTERIA ASSOCIATED WITH ATROPHIED TISSUES. 

The small bacillus above referred to (p. 195) varies greatly in size. The smallest 
(fig. 8, pi. xx) measure less than o.//; in thickness and 1.5/4 in length. The large ones 
(fig. 9, pi. xx) average 1.5/i in thickness and jfi in length. The former are homogeneous 
when stained. The latter frequently appear to have very conspicuous granules just 



200 BULLETIN OF THE BUREAU OF FISHERIES. 

inside the cell wall. These are probably artifacts. The older bacilli (fig. 9, pi. xx) taper 
at one end. They were at first taken for protozoan spores. These bacilli occur by 
thousands in and near degenerate epidermis and muscle tissue. It is not unusual to find 
them grouped in the form of the cell which they have completely destroyed. They are 
then of nearly uniform size (fig. 10, pi. xx); but between individuals of separate groups, 
there is often a great difference in size. They stain, as a rule, with methylene blue, 
gentian violet, toluidin blue, and Giemsa stain. Inside the host cells (fig. 6, pi. xx) and 
when first set free from them they stain, if at all, with great difficulty. This may no 
doubt be due to a zoogloeic condition. In smears, the stretching of this secretion causes 
the bacilli to be drawn into long parallel rows. The secretion then resembles elastic 
connective tissue fibers and the bacteria replace the connective tissue nuclei. At times 
the zoogloea is not noticeable. (Fig. S and 10.) 

To what extent toxins emanating from the short bacillus are the cause of the death 
and disintegration of the host tissues we can judge from the following facts: As already 
stated, this bacillus is not to be found throughout large areas of atrophied muscle and 
integument. If the toxin emanating by diffusion from a localized organism brought 
about the decadence of a tissue, one would expect the evidences of such decadence 
to indicate a uniform advance of said toxin in the same direction through a given tissue; 
but, as we have seen, the atrophy of muscle fibers is limited to a certain few in a large 
number of normal cells, or there may even be a few normal fibers extending through and 
far into a necrotic region. The same relations prevail more or less in the epidermis. 
If the short bacillus is to be regarded as a saprophyte, then some more virulent primary 
organism must be present. In the diseased gills the abundance of M. musculi and the 
extent of injury in its immediate presence point to the sporozoon as the primary agent. 
There are a few places in the gill tissue where the short bacillus is abundant, but, as 
would be expected of a saprophyte, in very degenerate tissue only. Such seems to be 
its relation to all the tissues. 

There are also tissues in which nothing but the long bacillus can be recognized as 
the agent of primary degeneration. While never abundant, it may be observed more 
frequently than the short bacillus in fresh smears of infected tissue. After about 24 hours 
the latter appear in clusters in the muscle fibers occupying excavations of regular ovoid 
contour. The long type occurs less frequently in tissues that are completely atrophied 
than in those which just begin to show signs of decadence. Fresh muscle, in the latter 
condition, may have the long bacilli more or less abundantly distributed under the 
sarcolemma, but never in compact groups, a condition which is characteristic of the 
short form. In sections, the long type has been encountered, one or two at a time, 
in muscle fibers at or near the region of advancing degeneration, and occupying irregular 
transverse clefts in the scarcoplasm (similar to those in fig. 4, pi. xx). But these cavities 
seem to be much too large to be considered the excavations of so few of these minute 
organisms. Their toxins may precede them and the transverse cleavage of the muscle 
fiber may be due to subsequent mechanical forces. On the other hand, the bacillus is 
quite as likely to creep into the crevices in the sarcoplasm as are the blood tissues 
(p. 198). Its presence is therefore not necessarily evidence that it is the cause of the 
crevices. In one stained smear, some of the muscle fibers of which are completely 
hypertrophied, the long bacillus is very abundant, especially in the connective tissue. 
There is no evidence of the admixture of fluid from purulent tissue such as is frequently 



SPOROZOON PARASITES OF FISHES. 20I 

common when the short bacillus occurs abundantly; nor are there any of the short 
bacilli. The normal striated fibers possess few if any of the germs and they seem to be 
numerous in proportion as the sarcoplasm is degenerate. These are not the conditions 
we would expect of a virulent parasite unless its primary attack is through the agency 
of a toxin. There is a second factor to be considered, however, inasmuch as numerous 
myxoplasms and autospores of M. ynnsciUi occur in some of the less decomposed por- 
tions of the same tissue. With the evidence at hand bearing upon the virulence of the 
two bacilli, the most natural conclusion is that the short bacillus is a saprophyte, that 
the long bacillus is either a facultative parasite upon the post tissues, which has been 
reduced in vigor by the Sporozoa already established therein, or perhaps a true parasite, 
in which case there are frequent double infections, the long bacillus and Myxosporidia 
together preparing the way for the saprophytic short bacillus. 

The long and short bacilli are easily distinguishable by their size, shape, and habits. 
The long bacillus is o.jfi in diameter and usually at least 2.5// long, but it may be 22/4 
long, without any noticeable increase in diameter. (Fig. 1 1, pi. xx.) They have tapered 
ends, especially those which have but recently divided. Sometimes the long type 
divides, forming short rods, but they are then in chains. They never occur in clusters 
as in figure 10, plate xx. The short type is never coiled, never so long, and always thicker 
than the long bacillus. They are both encountered in smears which include the fluids 
of completely broken-down tissues, but the short form is always abundant in such 
fluids, while the former is rare. One is frequently clustered and in regular pockets, the 
other isolated or scattered and, if in cavities at all, they are irregular crevices. 

SPOROZOA ASSOCIATED WITH ATROPHIED TISSUES. 

From the evidence in the foregoing pages and borne out by that which is to follow, 
it is certain that a sporozoon causes the primary degeneration of muscle, gill, and pos- 
sibly integumentary tissues, resulting in pathological conditions which are quite as 
characteristic as when the bacillus is the primary parasite. In one tissue which was 
sectioned (fig. 18, pi. xxi) the degeneration of the muscle fibers is identical to that where 
bacteria alone have been observed (p. 200). The atrophied fibers, which contain numerous 
scattered Sporozoa (p. 198, 203), occur in groups of two or three here and there through- 
out the fragment of flesh. Frequently, in both sections and smears, degenerate muscle 
fibers occur in which there are cells similar to the above but with neither nucleus nor 
cytoplasm stained ; also large amoeboid masses of granular cytoplasm without any visible 
nucleus (fig. 13, pi. xx). Usually such foreign cells occur in tissues when either myxo- 
spores or multiplicative stages are more or less abundant. 

In one or the other of the above stages the sporozoon has been positively identified 
with the disease in 18 of the 85 fish which have been examined. On the other hand, 
many degenerating fibers have been encountered both in smears and sections in which 
neither Protozoa nor bacteria could be found. In such cases there is about equal 
lack of evidence that either of the above are the causal agents of such disintegration. 
While it is probable that the majority of the sores are caused by the inoculation of a 
wound by a germ, there is less evidence of a primary attack upon the tissues by the 
bacteria, except through a widespread toxin, than by Sporozoa. In this connection there 
is probably a significant difference in the external appearance of diseased tissues which 
are primarily due to the sporozoon attack and those which are caused by bacteria. 



202 BULLETIN OF THE BUREAU OF FISHERIES. 

Certain fish in which the diseased parts were conspicuously congested (ventral part of 
the head, around the anus, and about the eyes) were almost invariably found to con- 
tain a large number of myxospores. When we consider the unknown stages of the 
Sporozoa which, according to the cyclic habit of these organisms, advance from stage 
to stage in a given culture at nearly the same rate, there is reason to attribute to them 
more destruction than our observations warrant. Our present lack of knowledge is no 
doubt due in part to the inadequate stains that have been employed and in part to the 
confusion of tissue cells with certain stages of the myxosporidian cycle. (See also p. 205.) 

STAGES OF MYXOBOLUS MUSCULI. 

Mention is made in the literature of but one other case of mxyosporidian disease 
of the integument and flesh which is closely allied to that of the Fundtdus, namely M. 
lintoni of Cyprinodon variegatus (Linton, 1 889-1 891). With this one exception, similar 
diseases in other American and European salt-water minnows, as far as we can learn, 
have not been described. The M. lintoni of the Cyprinodon was at first supposed to be 
identical to the M. musctUi of Fundulus. But very recently a tumor of the variegated 
minnow was encountered. (See p. 206.) Both the spore and the tumor are markedly 
different from the common condition of Fundulus. 

The myxoplasm of M. musculi produces a great many pansporoblasts, each with a 
single spore. There is a large vacuole in most of the spores which is the characteristic 
iodinophilous vacuole of the genus Myxobolus, to which the parasite undoubtedly belongs. 

Of the life history we have the spore, pansporoblast, possibly the m>TCoplasm, 
schizont, and multiplicative or autospore. In but 3 of the 18 fish which harbor Sporozoa 
have we stages (figs. 20, 21, 26, 27, pi. xxi) that can be unmistakably connected with the 
spore. By association in the same tissue or by the appearance and staining reaction 
we have probably identified the myxoplasms and autospores. 

According to Auerbach's (1910) description of M. bergense, the spore terminates 
the life cycle in a given host and starts a new cycle in a new host. We can but assume 
that the trophoplasm of M. musculi likewise arises in some way from a primary myxo- 
spore. The trophoplasm (fig. 12, pi. xx) is difficult to stain, and therefore its sporozoon 
properties are not always certain. (See also Chloromyxum properties, p. 205.) Spherical 
or oval spaces in the diseased myoplasm and in the epidermal cells (possibly identical, 
fig. 36, pi. XXI, and p. 197) are very abundant. These are probably multiplicative tropho- 
plasms, unless we have confused them with fat or other nonstaining substances. Some- 
times these bodies have nuclei (fig. 12) which, though usually faint, may stain deeply. 
It is not impossible that some of these small trophoplasms may be those of the Chloro- 
myxum. When large, the trophoplasms have a granular structure (fig. 13, pi. xx) and are 
doubtless preparing to undergo schizogony. We have encountered but five or six 
such schizonts. In one series of sections they are associated in diseased muscle fibers 
with cysts containing many spores. (Fig. 14, pi. xx.) The amoeboid form of the mature 
schizont is characteristic and distinguishes it from the smaller forms. The schizont 
in figure 13 is 33/1 wide by 74/4 in length. Some of the cysts are about this size, but 
figure 14, which is 19/4 wide and 24// long, is a section through the small end of a cyst 
of only moderate size. The cysts are found both within and between the muscle fibers. 
They contain several hundred spores, the nucleus of which, like that of the trophoplasm, 
has at times little affinity for the stains we have employed. The spores sometimes 



SPOROZOON PARASITES OF FISHES. 203 

appear to be spherical in form (fig. 14, pi. xx) and vary somewhat in size. They have 
a small faintly-staining nucleus and hyalin nonstaining cytoplasm. Isolated spores 
and masses of spores recently discharged from the cysts also occur in the smear prepa- 
rations associated with the intrafibrillar masses of material that appear to be equivalent 
to schizonts. These spores also occur in small numbers in the diseased gill where 
myxospores and sporoblasts are to be found in very great numbers. 

The occurrence of a multiplicative process of reproduction amongst the Myxo- 
sporidia in the manner here described is not uncommon. We have authentic cases 
in gall parasites of the flounder, and they have been described in M. pjeifjeri (Keys- 
selitz, 1908) and in Henneguya gigantea (Nemeczek, 191 1). While there is no question 
but that there are multiplicative spores, our evidence that the spore here described 
is such is, as with the trophoplasm, far from conclusive. Judging from the meager 
evidence at our disposal, there is about equal reason for considering it a young sporo- 
blast or a young trophoplast. It is more harmonious to regard these amceboid spores 
as the progenitors of both multiplicative and propagative trophoplasts and the oval 
spores, which are described below (p. 204) as sporoblasts, more especially since they 
apparently arise by free cell formation and in smaller numbers. 

The propagative and sporoblast stages have been encountered frequently in both 
sections and smear preparations. One series of sections of diseased integument and 
muscle (referred to on p. 198), which were cut approximately at right angles to the body 
surface, contains numerous large cells (fig. 18, pi. xxi) with small well-stained (with 
haematein) nuclei. Some of the muscle fibers are cut obliquely. They lie in the midst of 
healthy tissue and under integument which is apparently healthy. These myxoplasms 
are of oblong or spherical form with more or less even surface. The cytoplasm is 
tolerably homogeneous and does not retain the stains. The karyoplasm is also unstained, 
but the chromatin is somewhat conspicuous. These cells occur abundantly throughout 
60 or more sections, in 6 to 8 adjacent fibers, also in other distant fibers. Upwards 
of a hundred perfectly normal fibers around them have not a single foreign cell. Such 
cells are always intracellular. The sarcoplasm is considerably modified. The fibrillar 
structure is lost and the appearance is almost homogeneous. 

A cell similar in every respect to the myxoplasm just referred to, occurs abundantly 
in smears of muscle tissues. It stains less readily than do leucocytes and has smaller 
nuclei with less conspicuous chromatin. Myxospores and pansporoblasts have been 
found in their midst, in fact are to be found on slides where this type of cell occurs and 
not elsewhere. In this connection it is interesting, and perhaps additional evidence of 
relationship, that the same sore from which the sections containing these myxoplasms 
(fig. 18, pi. xx) were made also supplied a smear preparation containing numerous myxo- 
spores and pansporoblasts represented in figure 26, plate xxi. The sporoblast resembles 
the myxoplasm of smear preparations in shape, clear, nonstaining cytoplasm, size, and 
feebly staining (with methylene blue) nucleus. It is for the above reasons that this 
type is assumed to belong in the propagative cycle. 

There is a wide range of conditions to be seen in the nuclei of these myxoplasms, 
as well as some variations in size. Some densely-staining, cigar-shaped bodies (fig. 17, 
pi. xxi) almost devoid of protoplasm are embedded in the sarcoplasma, and others are 
closely applied to the myxoplasms (fig. 18, pi. xxi, near right-hand upper comer). The 



204 BULLETIN OF THE BUREAU OF FISHERIES. 

conditions suggest conjugation, but the stages are too few to indicate a succession of 
events. One myxoplasm contains two oblong spores. Elsewhere, replacing a degen- 
erated muscle fiber, are numerous small cysts (12// in diameter) with eccentric nuclei, 
which contain from four to ten or a dozen clearly defined oblong spores (fig. 16, pi. xx). 
These spores are found abundantly in other fish. (Fig. 19, pi. xxi.) They appear to 
arise by free cell formation. They are characterized by a transparency and a failure to 
stain that recall both the trophic stages and the sporoblasts. The nucleus, however, 
does stain faintly. It is quite large when the spores are set free. The latter measure 
4/i by 2.5/x and sometimes assume a spherical or amceboid form. Between this condi- 
tion and the mature sporoblast we lack recognizable connecting stages. They are 
not far removed, however, from the latter, which are spherical cells with very large 
nuclei. (Fig. 35, pi. xxi.) These occur in the gill above mentioned and have there been 
definitely connected with the myxospore. The pansporoblast has been encountered, 
along with spores and sporoblasts, in fresh smears of muscle. These are apparently 
identically homologous to those described for M. pfeifferi in the gills of the barbel 
(Keysselitz, 1908). If so, the sporogenesis there related would appropriately apply to 
M. musculi. INIany stages in the genesis of the spore are represented in one of our 
smear preparations. These have propagative stages (Keysselitz, 1908) as follows: 
First the sporoblast with large nucleus (fig. 35, pi. xxi) and two-parted pansporoblast 
(sporocyst) (fig. 22, 23, pi. xxi), which, according to Keysselitz, arises after a process of 
autogamous conjugation. The sporocyst apparently sets free the sporocytes before 
sporogenesis has proceeded far (fig. 21, pi. xxi). Giemsa stain does not reveal all the 
nuclei concerned in sporogenesis. Valve cells are formed (fig. 24, pi. xxi) before the 
polar capsules appear as large spherical bodies (fig. 25, pi. xxi). Later the myxospore 
becomes elongated and tapered (fig. 20, 26, pi. xxi). Two preparations have multitudes 
of immature spores. They are all free from the sporocyst protoplasm and have thick 
valves. It is therefore rather perplexing to explain figures 20 and 26. Perhaps the 
spore is about to be discharged in figure 20. Considerable variation in this respect 
occurs amongst some of the gall Myxosporidia. 

There are myxospores in 12 of the 85 fish examined. In but 3 of these do they 
occur in great numbers. With two exceptions (in diseased gills), the myxospores are 
not assembled in a manner that would suggest their origin from cysts or masses of 
pansporoblasts, as is common in other species of Myxosporidia. The two cases referred 
to may not be interpreted as evidence of this condition, but rather that the pansporo- 
blasts, where very numerous, have been packed close together. There are at least a 
thousand well-stained spores in the preser\'ed tissues. Not one occurs in the 10 tissues 
of which sections have been made. But those same tissues which contain spores have 
supplied all the propagative myxoplasms. 

The myxospores are very small (fig. 28, 29, pi. xxi). They average 14.3/t in length 
and 6.7/1 in width. In one fixed individual the plane at right angles to that passing 
through the polar capsules is presented. It measures 6.7/1 in thickness, from which 
we conclude that they are approximately circular in section. But another fresh spore 
was flattened in a plane perpendicular to that of the polar capsules and sutures to about 
two-thirds its width (fig. 30, pi. xxi). The polar capsules of myxospores average 6.5// in 
length and 2/1 in thickness. When extruded the filament is three to four times the length 
of the spore (fig. 29, pi. xxi). Coiled within the polar capsule, the filament makes from 



SPOROZOON PARASITES OF FISHES. 205 

10 to 14 turns (fig. 26, 28, pi. XXI). In young spores the valves are quite thick and may 
be seen at the edges as a pale border to the spore, but in mature spores they are thin 
and almost invisible. Young spores are shorter (i2u) and wider (7.5/i) than the 
mature spores and the polar capsules are not so long (6,u). They lengthen out as they 
approach maturity. When young, the nuclei stain with great difficulty, if at all. The 
sporoplasm occupies all the space at the large end of the spore. A large vacuole is nearly 
always visible in the sporoplasm. There are also dense areas and from i to 10 nuclei. 
(Fig. 20, 28, pi. XXI.) The nuclei are unstained in figure 29, plate XXI. There are some- 
times seven greenish-blue nuclei (fig. 28, pi. xxi) and three rather irregular dark-blue 
bodies between the polar capsules. It is not possible to be sure that this (10) is the 
maximum number as some of these are ill defined. A number of spores have their 
nuclei attached near the large end of each polar capsule, thus identifying them as the 
" polar capsule" nuclei. Probably the remaining four belong to the sporoplasm. It is 
not possible to recognize the "wall nuclei" at this stage, and the "resting nuclei" of 
the pansporoblast are doubtless lost. 

CHLOROMYXUM FUNDULI. 

The Chloromyxa which have been observed in the muscle of other fish (p. 208) are not 
identical to that found in Fuiidulus. C. quadratiim, which resembles the latter, has 
myxospores measuring 6/; in diameter by 5/i along the polar axis, while the Chloro- 
viyxuni of the Fundidus measures 7.5// in diameter and 6// along the polar axis and 
dififers in shape. The spore of C. quadratiim, when seen in line with the polar axis, has 
the sides deeply concave, and in the other plane it is more pointed. The polar capsules 
are also much shorter. They also differ in the relation of the spore to the pansporoblast 
and in the pathological effects. (For description of spore of C. fundiili see p. 208.) No 
reference to a myxospore of this character has been found by the writer. The name 
C. junduli has therefore been applied to this species. 

The myxospores of C. /M«<f!(/f have been encountered in but one fish. If the mv-xo- 
plasm occurs in other preparations, it has not been possible to identify it, although many 
suspected myxoplasms exist. It is not very probable that they are at all uncommon. 
They do not take up a particle of such stains as we have emploj'ed. The single slide 
containing this species is a smear preparation made from the diseased flesh of a fish 
which died in jar no. i of the experiment reported on page 196. It is stained with 
Giemsa stain. 

The muscle of this fish is in an advanced stage of decomposition. When the fresh 
slides were examined no myxospores were noticed, being difficult to see without a stain, 
but the sporoblasts were observ^ed without recognizing their importance. 

Bacteria are present on the slide but lacking in the muscle fibers. The decadence 
of the muscle must in this case be ascribed to the Chloromyxum, which is abundant in 
the hypertrophied muscle. The muscle is full of cavities containing unstained mjoco- 
plasms and sporoblasts which are identical in appearance to those of many other prepara- 
tions of diseased Funduliis. While this case introduces the possibility that manj' of the 
Fundulus cancers may be caused by Chloromyxum funduli, it gives very substantial sup- 
port to the agency of Sporozoa as the cause of these diseases. Since Chloromyxum and 
Myxobolus are not uncommon in muscle tissue, double infections are to be expected. 
But having failed to encounter myxospores of the Chloromyxum in over 100 stained 



2o6 BULLETIN OF THE BUREAU OF FISHERIES. 

preparations that have been examined, we are inclined to consider the Myxoholus more 
abundant and therefore the more common causal agent. 

The myxospore of C. funduli is about 7.5;/ in diameter, with a polar axis somewhat 
shorter (6//). At right angles to the polar axis, it is circular. There are four polar 
capsules, which taper to the apex of the spore, curving so as to conform to the constric- 
tion of the spore, which provides it with a blunt pointed apex (fig. 31 , 34, pi. xxi). There 
are four conspicuous nuclei (black), one near the base of each polar capsule. The sporo- 
plasm is stained a pale blue by the Giemsa. The polar capsules do not stain (fig. 31, 34, 
pi. XXI) . There are occasional myxospores of considerable size to be seen inside the sporo- 
blasts when the latter do not take up a particle of stain. Such clear hyalin amoeboid 
pansporoblasts are numerous throughout the sarcoplasm. They vary in size from a 
diameter of about 211 to four or five times the diameter of the spore. 

PROTOZOA RELATED TO THOSE HERE DESCRIBED. 

Numerous Myxosporidia parasitic upon either integument, gill epithelium, con- 
nective tissue, or muscle of fish have been described by other authors. About most of 
them we have very meager information. M. lintoni (Linton, 1889; Gurley, 1893) of 
Cyprinodon variegatus (short minnow), as already stated (p. 202), more closely resembles 
the parasites of Fundulus than any other species of which we know. The difference 
at first seemed to be slight and to be easily accounted for by a difference in the age 
of the spores. But when a case of the Cyprinodon tumor was finally obtained and 
examined, the indentity of the parasites in the two hosts, as well as the nature of 
the lesions, was found to be different. The "irregular fungoid elevations," described 
and figured by Linton and observed again by the writer, are of the nature of cysts con- 
taining spores, located in the integument, whereas the elevated scales in Fundulus are 
due to an infection of the epidermis by bacilli and a subdermal atrophy of the muscle. 
No tumor or spore-filled cyst has ever been encoimtered. The Cyprinodon tumor which 
we examined developed in a comparatively short time, probably less than a week, 
though the period can not be accurately stated. It caused the death of the fish the day 
following that on which it was first noticed. After the death of the host, the tumor was 
8 mm. wide by 10 mm. long, and caused a conspicuous elevation from the back of the 
fish anterior to the dorsal fin, about 2 to 2% mm. thick. It was of a j'ellowish-pink color 
when seen through the slightly pigmented integument. The scales were practically 
undisturbed and the integument was completely intact, in this respect differing remark- 
ably from the Fundulus sores. Beneath the tumor, the flesh contained intrafibrillar 
myxoplasms and sporoblasts with occasional spores, while the tumor itself was almost 
wholly a mass of myxospores, the latter numbering millions. We have already described 
(p. 197) a totally different condition in the Fundulus, resulting from the M. musculi. 

There is such a difference in the appearance (Linton, 1889, fig. 3) of the spores that 
they are readily distinguished. One can not be certain, however, that such differences 
are not due to the comparison of different stages in the development of spores of the same 
species. We have shown that the spores of M. musculi grow longer as they mature and 
the spore wall becomes thinner (p. 205). This fact would explain in part the discrepancy 
in the dimensions of the spores from the Fundulus and Cyprinodon. But, since the 
Spore of M. lintoni measures 13.9/t in length, i ip. in width, and 8// in thickness (at right 



SPOROZOON PARASITES OF FISHES. 207 

angles to the planes of the two polar capsules), and the mature spore of M. musculi 
measures 14.3/i in length by 6.7// thick, and from 4 to 6.7/i in width (see p. 204), in indi- 
viduals of apparently the same stage of development, it still seems that a sufficient 
discrepancy in size exists to supplement the marked differences in the pathological con- 
ditions. It may yet prove that the latter are due to the influence of different hosts, inas- 
much as we have one case of a Fundulus with a typical M. musculi lesion, but having 
spores indistinguishable from those of M. lintoni in either size or appearance. 

M. lintoni presents another contrast to the conditions in Fwndulus. In the former, 
calcareous bodies were observed amongst the spores by Linton (1889) and the writer, 
whereas nothing of the kind has ever been encountered in the hundreds of Fundulus 
tissues which we hav^e examined. 

Although the name M. lintoni was for a time retained for the Fundulus parasite, the 
present state of our knowledge will not permit of this assumption. The species " musculi" 
has been adopted because of the interesting and characteristic attack which the tropho- 
plasm makes upon muscle fibers. 

The spore of Myxoholus oviformis (Thelohan) resembles M. musculi very much in 
appearance, but is less tapered and shorter (Thelohan, 1894). 

The following, for one reason or another, are also of interest in their bearing upon 
M. musculi. A "Myxosporidian" of unknown genus and species was found by Linton 
(1899) in the connective tissue of the entire body of Notropis megalops Rafinesque 
(albeolus Jordan), the shiner. The epidermis is marked by dark purplish blotches. The 
scales are absent in most cases. A "Myxosporidian" of unknown genus and species was 
observed by Lieberkiihn (1854) in the connective tissue of Gasiereosteus aculeaius (stickle- 
back). The skin is said to have contained cysts. The conditions seem to be unlike those 
in Furuiulus. Cyprinus leuciscus (Muller, 1841) has been observed with tumors in the 
integument caused by a species of Myxobolus. M. oblongatus Gurley produces cysts 
under the scaleless skin of the head region in Catostomus tuberculatus Le Sueur (Gurley, 
1891, 1893, p. 234). M. transovalis Gurley (1893) of Phoxinus (Clinosiomus) funduloides 
Girard, occurs under the scales and external to the epidermis. "It forms a thin dis- 
coidal mass situated in the center of the concave undersurface of the scale." That it 
is not identical with M. musculi is certain from the dimensions of the myxospore (length 
6/(, breadth 8/(), the diameter of which, at right angles to the polar axis, is greater than 
through the polar axis. We have very scanty information concerning the M. strongylurus 
(Gurley, 1893, p. 247), which is found encysted in the skin of the head of Synodontis 
schal; of M. momiirus (Gurley, 1891 , p. 416), known from cysts in the subcutaneous inter- 
muscular tissue of Aphrcdodcrus sayanus Gilliams; of Henneguya nusslini Schuberg und 
Schroder (Leger, 1906), which is found in the connective tissue of the dorsal fin of the 
trout; of M. gigas Auerbach (1907), which thickens the integument at the ventral angle 
of the gill in Abramis brama Linnaeus (bream); and of a Myxobolus of unknown species 
described by Borne in 1886 (Gurley, 1893, p. 244), which causes great tumors over the 
surface of Leuciscus rutilis. 

In Coregonus jcra there occurs a common disease of the integument caused by a 
species (M. zschokkei, Gurley, 1893; Zschokkei, 1884), the myxoplasm of which is not 
known. The cysts lie in the subcutaneous connective tissue and between the muscles. 
It causes irregular thick patches on the skin, from which the scales drop. 



2o8 BULLETIN OF THE BUREAU OF FISHERIES. 

PREVALENCE OF MYXOSPORIDIAN INFECTION IN RSH. 

The infection of muscle tissue by Myxosporidia is quite common in fish. A parasite 
belonging to the genus Chloromyxum occurs in the flesh of the young herring and young 
alewife (Linton, 1891). Both the pansporoblast and spores of a Chloromyxum have been 
found abundantly by the writer inside the fibers, and the spores also assembled else- 
where in large cysts. A fuller account of this species will be published later. The 
muscle cells of Callionymus lyra are also subject to an intracellular parasite (Glugea 
destruens Thelohan, 1891; Henneguy et Thelohan, 1892; Gurley, 1893), the myxoplasm 
of which has not been observed. It causes the muscle fibers to undergo degeneration. 
Chloromyxum quadratum (Thelohan, 1894) also occurs in the muscles of this fish. It is 
also reported in the flesh of Coris julis, Syngnaihus acits, Trachurus Irachurus (Minchin, 
1903) and Nerophis aquoris. In Coitus scorpio the muscle tissue is attacked by Pleisto- 
phora typkalis (Thelohan, 1890, and 1891 ; Gurley, 1893). Both pansporoblast and spores 
have been found, but they are intercellular in position. The muscle fibers are displaced 
but do not degenerate. Leptotheca perlaia (Gurley) occurs in the muscles of Acerina 
cernua Linnseus. Of these species there are none that closely resemble M. musculi. 
Numerous cases of Myxoboli are known to inhabit gill tissues. Auerbach (191 1) lists 
22 species of Myxobolus which have been described in the gills of fish. But we have 
encountered nothing that might be considered identical to M. musculi. 

The disease of Fundulus is remarkably like that which has so frequently caused 
epidemics amongst the barbel {Barbus barbus Linnaeus) of European rivers. The latter 
is caused by M. pfeifjeri Thelohan (Raillet, 1890; Ludwig, 1888; Thelohan, 1894). It 
produces both tumors and ulcers and occurs encysted and free in muscle, liver, kidney, 
spleen, and connective tissue. The tumor when formed does not at all times break 
through, either into the body cavity or to the outside. It is not an integumentary 
parasite at the beginning as those of Fundulus seem to be. The tumor commonly occurs 
amongst the connective tissue and the muscles of the body wall. The parasite may be 
encysted in a thin restraining membrane produced by the host. Numerous indi\aduals 
of about the same age tend to gather in groups and become isolated in tube-like cysts. 
The muscle fiber is invaded and undergoes a "vitreous alteration" (Thelohan, 1893) 
leaving "yellow granulations as degeneration products" (Keysselitz, 1908). Thelohan's 
figure 5, plate vii (Thelohan, 1894), representing a muscle fiber containing myxoplasms 
in transverse crevices recalls, very vividly the appearances we have encountered in the 
degenerate muscle of Fundtdus (fig. 4, pi. xx). The tumors may soften and become a 
"stinking abscess containing spores" (Ludwig). M. pfeifjeri passes through distinct 
cycles of development which is no doubt the case in M. musculi. In April it is in a vege- 
tative stage in which the multiplicative reproduction prevails; later propagative repro- 
duction is encountered and myxospores are developed. The rate of advance of the dis- 
ease depends upon the temperature (Keysselitz). 

Both Keysselitz and Thelohan describe bacteria in tissues of diseased barbel. 
Keysselitz says bacteria contribute liberally to the formation of the tumors. These 
bacilli are found only in the tissues infected by Myxosporidia. They prevent the 
growth of connective tissue and bring about degeneration (gangrene) of the tissue. 
These bacilli are "as long as the spore" (PfeifFer, 1890) (6/(, Thelohan) and stain easily 
with methylene blue and gentian violet. (This is also true of the bacilli of Fundulus 



SPOROZOON PARASITES OP FISHES. 209 

diseases.) Pfeiffer mentions threads attached to these bacilli. A coccus is also occa- 
sionally found. The presence of bacteria is therefore not necessarily an indication that 
they are primary as causal agents of disease since M. pjeifferi is known to be the cause 

of the barbel disease. 

GENERAL CONCLUSIONS. 

L The sores of Fundulus are usually caused primarily by lesions. These may 
occasionally be due to parasites such as leeches, distomes, and copepods, but usually 
to rough handling and carnivorous enemies. 

n. At least four kinds of germs invade these lesions and bring about hypertrophy 
of the tissue elements and decomposition, namely, two species of bacteria and two species 
of Myxosporidia. 

in. There is doubt as to the virulence of the bacteria. One species at least is 
saprophytic. There is no doubt as to the virulence of the Myxosporidia when present. 

IV. Cleanliness, careful feeding, and aeration bring about recovery in practically 
all injured fish. It can not be claimed that fish which are known to have Myxosporidia 
are curable. 

V. The trophoplasm of both species of Myxosporidia attacks the muscle fibers, 
that of the M. musculi also attacks the gill connective tissue. 

VI. Blood elements, especially nuclei, give rise to abundant artifacts which are 
closely associated with the parasite involved. 

VII. Sporogenesis of the Myxobolus occur infrequently in the muscle and gill tissues. 

VIII. Multiplicative spores are probably formed in M. musculi in addition to pri- 
mary sporocytes. 

IX. The myxoplasm of both C. jundidi and M. musculi are stained with diflSculty 
and are therefore not easily found. 



BIBLIOGRAPHY. 

AuERBACH, Max. 

1907. Weitere Mitteilungen iiber Myxobolus Kglefini Auerbach. Zoologischer Anzeiger, bd. 

XXXI, p. 386-391. Leipsig. 
igio. Biologische xmd moqjhologische Bemerkungen uber Myxosporidien. Zoologischer Anzeiger, 

bd. XXXV, p. 57-64. Leipsig. 
igii. Unsere heutigen Kenntnisse iiber die geographische Verbreitung der Myxosporidien. 
Zoologischer Jahrbucher, Abtheilung fiir Systematik, bd. 30, p. 471-494. 
Borne, Max von dem. 

1886. Handbuch der Fischzucht und Fischerei, p. 211, fig. 215. 
GURLEY, R. R. 

1891. On the classification of the Myxosporidia, a group of protozoan parasites infesting fishes. 

Bulletin United States Fish Commission, vol. xi, 1S91, p. 407-420. Review in Central- 
blatt fiir Bakteriologie und Parasitenkunde, bd. 15, p. 86-88. 
1893. The Mjrxosporidia or psorosperms of fishes, and the epidemics produced by them. Report 
U. S. Commission of Fish and Fisheries, 1892, p. 65-304, pi. 1-47. 
Henneguy, F., et Thelohan, P. 

1892. Sur im Sporozoaire parasite des muscles de I'ecrevisse. Comptes rend us hebdomadaire 

Soci^td de Bioiogie Paris, t. 4, p. 748-749. 
Keysseutz, G. 

1908. Die Entwicklung von Myxobolus pfeifferi Thelohan, Theil i imd 2, Archiv fiir Protisten- 

kunde, bd. 11, p. 252, 276-308. Jena. 
Leger, L. 

igo6. Sur une nouvelle myxosporidian de la tauche commune et de la truite indigene. Comptes 
rendus de I'Academie des Sciences, Paris, t. 142, p. 655. 

LlEBERKUHN, N. 

1854. ijberdie Psorospermien. MuUer's Archiv, p. 9-10, 22, 24, 354, taf. 2, fig. 28; taf. 14, fig. 9-12. 
Linton, Edwin. 

1889-1891 . On certain wart-like excrescences occurring on the short minnow, Cyprinodon variegatus, 
due to psorosperms. Bulletin United States Fish Commission, vol. ix, 1889, p. 99-102, 
pi. xxxv; ibid. p. 359-361, pi. cxx, fig. 1-3. Review in Centralblatt fiir Bakteriologie 
und Parasitenkunde, 1892, bd. 11, p. 475. 

LUDWIG, H. 

1888. tjber die Myxosporidien krankheit der Barben in der Mosel. Jahrbuch des rheinischen 
Fischerei Vereins, Boim, p. 27-36. 
MiNCHIN, E. A. 

1903. A Treatise on Zoology. Edited by E. Ray Lankester, pt. i, fasc. 2, p. 150-361. 
MtJLLER, J. 

1841. Ueber Psorospermien. Miiller's Archiv fiir Anatomic und Physiologic, p. 477-496, pi. 16. 
Abstract in Microscopical Journal, London, 1841-2, p. 123-124. 
Pfeiffer, L. 

1890. Die Protozoen als Krankheitserreger, Jena, i ed. 
Plehn, M. 

1905. ijber die Drehkrankheit der Salmoniden (Lentospora cerebralis) Archiv fur Protistenkunde, 
bd. 5, p. 145-166, taf. I, fig. 7. Jena. 
RaillET, M. a. 

1890. La maladie des Barbeaux de la Mame. Bulletin Societe Centrale d'Aquiculture, Paris, 

t. 2, p. 117-120. 
210 



SPOROZOON PAR.'iSlTES OF FISHES. 211 

Thelohan, p. 

iSgo. Contribution a 1 '^tude des Myxosporidies. Annales de Micrographie specialement consacrees 

a la bacteriologie, aux protophytes et aux protozoaires. P. i, t. 2, p. 193-213. Paris. 
i8go. Recherches sur le developpement des spores chez les Myxosporidies. Comptes rendus heb- 

domadaire de la Society de Biologic. Paris, t. 2, p. 602-604. Abstract in Journal Royal 

Microscopical Society, 1890, pt. 2, p. 194-195. 
1891. Sur deux sporozoaires nouveaux parasites des muscles des poissons. Ibid., t. 62, p. 168-172. 

1893. Alterations du tissu musclaire dues a la presence de Myxosporidies et de microbes chez le 

barbeau. Ibid., t. 5, p. 267-270. Abstract in Centralblatt fiir Bakteriologie und Para- 
sitenkunde, bd. 14, p. 532. 

1894. Recherches sur les Myxosporidies. Bulletin Scientifique de la France et de la Belgique, 

t. 26, p. 100-394, pi. 7-9. Paris. 
WlERZEJSKl, A. 

1898. tjber Myxosporidien des Karpfens. Anzieger der Akademie der Wissenchaft in Krakau, 
Marz. R^sum^ in Bulletin International de I'Academie des Sciences de Cracovie, Comptes 
rendus des seances, 1898, p. 129-145. Cracovie. 
ZSCHOKKE, F. 

1884. Psorosperms de Coregonus fera, Archives de Biologie, t. 5, p. 234-235, pi. 10. 



EXPLANATION OF PLATES. 

With the exception of figures 20, 27, 30, and 35, the drawings were made with the 
aid of a camera lucida. For figures 3, 5 to 11, 15 to 17, 19, 21 to 26, 28, 29, 31 to 34, and 
36 a no. 12 Bausch & Lomb compensating ocular and one-twelfth inch oil immersion 
objective were used. For figures i, 2, 12, 14, and 18 a Bausch & Lomb i-inch occular 
was employed with the same objective. The i-inch occular and a one-fifth inch objec- 
tive were used in figures 4 and 13. All figures have been reduced to two-thirds the size 
of the camera images. The tube length was 160 mm. and the camera arm 90 mm. 

The figures are numbered approximately in the order of development. Figures 2, 

3, 5, 6, 7, 8, 9, and 10 are made from the same slide, and figures 13, 14, 16, 17, 18, 26, 

and 28 are from the same fish. 

Plate XX. 

Fig. I. A bit of infected muscle from a smear of a sore on the side of a small Fundulus hetercliliis in 
the first stage of disintegration. Fixed in corrosive sublimate and acetic acid and stained with Mayer's 
haematein. The pale bands of the fiber are beginning to become granular at one end. Fibrin threads 
have been spread over it in making the smear preparation. ( X860.) 

Fig. 2. A bit of degenerating muscle fiber. Numerous artifacts and a degenerate erythrocyte 
nucleus occur in the sarcoplasm. The granular striae are degenerated sarcolymph. Note the sarco- 
plasm is also becoming granular. ( X860.) 

Fig. 3. From a smear of a bit of degenerating muscle in a sore on the side of Fundulus majalis. 
The integument more or less disintegrated, scales entirely absent. Fixed in absolute alcohol, ether, 
and formaldehyde. Stained in methylene blue, orange G, and eosin. Sarcous elements have lost 
their sharp rectangular form and are becoming granular. A characteristic muscle artifact is distributed 
between the sarcostyles and some are just beginning to become amoeboid in form. (X2000.) 

Fig. 4. A characteristic appearance of a degenerating muscle fiber which may or may not be a later 
stage than those represented in figiyes 2 and 3. Neither bacteria nor Myxosporidia are necessarily 
present in these spaces. Both have been encountered there. (X400.) 

Fig. 5. A fragment of degenerating muscle upon and into which erthrocytes and leucocytes have 
entered. The cytoplasm of the latter is disintegrated and the nuclei are in an advanced stage of 
degeneration. (X2000.) 

Fig. 6. Atypical mass of degenerate nuclei containing unstained bodies which are probably zooglcea 
containing the short bacillus. There are cords of this material in which the bacilli are faintly visible. 
Such white areas are not merely transparent spaces but thick masses with stainable protoplasm above 
or below. (X2000.) 

Fig. 7. Artifacts from decomposing muscle fibers. In fresh muscle these are common after 10 to 
12 hours, appearing first between the sarcostyles. Older stages assume a more compact form. (See 
figures 3 and 2 . ) The stain is a homogeneous pale blue. Maximiim length 8. g/i. (X2000.) 

Fig. 8. The short bacillus. An isolated group near which are located cells containing white oval- 
shaped bodies like those in figure 6. Note the variation in size and shape. That one near the " X" 
sign measures 1.5/1 by 7.4/1; that near the " -|-" sign measiu-es 1.8/1 by i.i/i. (X2000.) (See also fig. 10.) 

Fig. 9. Short bacillus older than figure 8. Nearly the maximum size. Note the taper toward 
one end and the stainable granules. The latter are probably artifacts. Left-hand upper one measures 
5.2/1 by 1.4/1. (X2000.) 

Fig. 10. A cluster of long bacilli which have caused the complete breakdown of a tissue cell and 
rest in situ. (X2000.) 

Fig. II. Several of the long type of bacilli which are located just under the sarcolcmma of a muscle 
fiber that shows the first signs of degeneration. The small individual in the middle below has dimen- 
sions as follows: Length, 4.8/1; thickness, o. 7/1. (X2000.) 
212 



SPOROZOON PARASITES OF FISHES. 21 3 

Fig. 12. A section cut diagonally through a muscle fiber. This fiber is adjacent to the dermis. 
On the inner side the sarcoplasm is hypertrophied, on the outer side it retains the fibrillation. The 
oval bodies are interpreted as trophoplasms of the M. musculi. The large one has several spherical 
bodies which take a deep haematein stain, presumably nuclei. (X800.) 

Fig. 13. A muscle fiber in which there are the first evidences of disintegration. It contains two 
or more large trophoplasts or schizonts. The appearance of the cytoplasm is like that of other 
stages, pale and unstained, there being no sign of the nucleus. There is evidence of a complex system 
of pseudopodial extensions of the cytoplasm which is characteristic of the Myxosporidia. Large indi- 
vidual 84.7/1 by 193. 5/1. (X400.) 

Fig. 14. Multiplicative spores of M. musculi, presumably derived from a large trophoplasm such 
as figure 13. There is no cyst wall. In adjacent sections are fragments of the schizont nuclei mingled 
with the spores. The spores stain feebly with eosin and orange G. The nuclei are not stained deeply. 
19. 3/( in diameter. (X860.) 

Fig. 15. A myxoplasra of M. musculi in muscle from a smear preparation fixed with absolute alcohol 
and ether and stained with methylene blue. One side overlies a nucleus of the muscle fiber. The 
pale bands of the muscle fiber may be seen. The muscle stained deeply and the parasite pale. The 
protoplasm is finely granular and there is only a suggestion of a cytoplasmic network. The nucleus is 
vaguely stained. i3.4;( by i8.6/(. (X2000.) 

Fig. 16. Formation of sporoblasts of M. musculi. This cyst is one of a mass numbering several 
hundred which occupy a position where a muscle fiber has been completely destroyed. The 10 spores 
stain very feebly. They lie in slight cavities of the protoplasm. Diameter of cyst la/c length of 
spore 4/(. (X2000.) 

Plate XXI. 

Fig. 17. A possible microgamete of M. musculi from amongst the numerous myxoplasms of muscle 
fibers adjacent to that shown in figure 18. The motile shape of several such structures, the small 
amount of cytoplasm, and close approximation to some of the large mj-xoplasms are noteworthy. (See 
right-hand upper region of fig. 18.) 6. 5/1 by 2.2/1. (X2000.) 

Fig. 18. A section of a muscle fiber of Fundulus heteroclitus cut crosswise at a slight angle. The 
scales in the region of this infection had dropped off, and the area was almost white, being slightly 
discolored by blood. The tissue was fixed in corrosive sublimate and acetic acid and stained first in 
Mayer's haematein, then in methylene blue, later in eosin and orange G. One of the structures in the 
sarcoplasm, that to the left in the middle, is the nucleus of a muscle fiber. The others are stages in 
the propagative cycle of M. musculi, primary and secondary sporoblasts. The large one in the middle, 
at the top, is 12. 6/1 in length and 5.9/1 in width. (X860.) 

Fig. 19. Three young sporoblasts of M. musculi from the smaller type of cysts represented in 
figure 16, plate xx. Note the increase in the size of the nuclei. They are typically free from cyto- 
plasmic stain. (See C./««(/«/i, fig. 31.) Lower individual 4« by 2.5/1. (X2000.) 

Fig. 20. A fresh sporoblast of M. musculi containing a spore which is almost mature. From a deep 
cavity in the flesh back of the head. Interesting in connection with figure 26. (Free-hand drawing, 
not to scale.) 

Fig. 21. Sporocyte of M. musculi expelled from pansporoblast. It forms the first st^e in the 
series represented by figures 23, 24, and 25. The nucleus is small and faintly stained, as is the rest of 
the cytoplasm. It has no external envelope. Diameter 11. 9/1. (X2000.) 

Fig. 22. A pansporoblast of M. musculi (sporocyst) with t%vo daughter cells, the nuclei of which 
are undergoing autogamous conjugation. ( X2000.) 

Fig. 23. A pansporoblast of M. musculi after the autogamous conjugation and subsequent division 
of the nuclei. 

Fig. 24. A sporocyst of M. musculi which has been set free from the pansporoblast. Apparently 
the sporoplasm remains attached to one myxospore (fig. 20), and the other is almost devoid of external 
protoplasm. The two wall cells are clearly visible, but without nuclei. The capsule nuclei are prob- 
ably formed but do not stain. One of the 12 nuclei happens to be in a suitable condition to take the stain. 
1 1. 9/1 by 13.4/1. (X2000.) 

Fig 25. A myxospore of M. musculi with a remnant of protoplasm. Two polar capsules are 
beginning to form. (X2000.) 



214 BULLETIN OF THE BUREAU OF FISHERIES. 

Fig. 26. A sporocyst of M. musculi from a smear of diseased integument of the mouth and head 
in front of the eyes. Elsewhere the sporocysts have less cytoplasm. It is the only one encountered 
in this condition. The failure of the nuclei to take the stain is characteristic. The myxospore is 
immature, being less slender than older myxospores. The details of the polar capsules are very trans- 
parent and stain dark blue, while the spore wall is a very pale blue. The vacuole and sporoplasm are 
prominent, but the nuclei of the spore can not be clearly discerned. Sporocyst, 17. 8/1 by 23.8/1; spore, 
14.8/1 by 7.4/1; polar capsule, 7.4/1 by 2.2/1. There are 13-14 spirals in the filament. Fixation; Absolute 
alcohol, ether, corrosive sublimate, acetic acid. Stain: Mayer's haematein, methylene blue, orange G., 
eosin. (X2000.) 

Fig. 27. A sporoblast of M. musculi from a fresh smear of degenerated muscle taken from a deep 
cavity (the same as fig. 20). Easily distinguished from tissue cells by the three nuclei. Protoplasm 
contains much coarsely granular matter. (Drawn free-hand, not to scale.) 

Fig. 28. Myxospore from the same slide as figure 26. The mature spore, when compared with that 
in the pansporoblast, is longer and more pointed at the polar end. The vacuole is probably an iodi- 
nophilous structure. The coiled filaments make 11 to 12 turns. The polar capsule wall is visible, but 
the spore wall can not be clearly seen. The valves and sutures are also indistinguishable. While there 
are as many as 12 blue and green bodies present, one can not be sure that all of them are nuclei. Seven 
or eight bodies are moderately conspicuous. Two lie in the wall of the polar capsules and are doubtless 
the capsule nuclei. 14.8/1 by 6.2/1. (X2000.) 

Fig. 29. A myxospore of M. musculi from large sores on each side of the tail of a Fundulus heteroclilus, 
caudal fin entirely gone. Fixed in absolute alcohol and ether, stained with methylene blue. Six 
unstained nuclei in the sporoplasm and one large vacuole. Filament discharged. Spore, 7.4/1 by 16.4/1. 
Polar capsule, 2. 2/t by 7.4/1. (X2000.) 

Fig. 30. Diagram of the cross section of a fresh myxospore of M. musculi as if seen from the end. 
The specimen was lying so as to present the edge of the valves to view. It is obviously flattened. The 
polar capsules also appeared to be, but one can not be certain about this. The sutures are straight and 
symmetrical. Fixation: Alcohol, ether, formalin; Giemsa stain. (This drawing not made to scale.) 

[Figures 31 to 34 are all from the same smear preparation of diseased muscle from a dead fish, being 
one of those taken from jar no. i (see pp. 195, 196).] 

Fig. 31. Pansporoblast of Chloromyxum funduli embedded in a degenerated muscle fiber. The 
contained myxospore has taken up the stain, but the protoplasm of the pansporoblast is absolutely 
devoid of visible structure. Note the even contour of the characteristic lobose pseudopodia. 15.2/1 by 
12/1. (X2000.) 

Fig. 32. One of a group consisting of free young myxospores of C. funduli. Like the mature 
myxospores, they stain readily, but their nuclei are not differentiated. They are, as a rule, not quite 
so irregular, but the pseudopodia are always small and angular. Note the contrast between these and 
the pansporoblasts. 3.7/t by 4.5/1. (X2000.) 

Fig. ^i- Myxospore of C. funduli. The outline is approximately circular. The sporoplasm is 
homogeneous but dense arotmd the four polar capsules, doubtless because of the greater thickness at 
this point. The four nuclei are always associated with the polar capsules, hence are doubtless capsule 
nuclei. Diameter, 8.g/i. (X2000.) 

Fig. 34. Myxospore of C. funduli seen from the side. Note the sporoplasm is not much denser 
about the polar capsules. The sporoplasm tapers to a blunt apex. In many it is more pointed. The 
polar capsules have long, curved, tapering necks with the large ends far apart. The capsule nuclei 
alone stain. 8.2/1 by 6.7/1. (X2000.) 

Fig. 35. A fresh sporoblast of M. musculi from the same slide as figures 20 and 27. The cyto- 
plasm is rich in granules. The nucleus is very large and has a conspicuous karyosome. (Not drawn 
to scale.) 

Fig. 36. An isolated epidermal cell derived from a mass near the margin of an advanced ulcer, 
most of which have numerous unstained bodies like those in the muscle fibers (fig. 12, pi. xx). From 
sections. The epidermal cell is not typical in appearance, but the unstained bodies are, and are iden- 
tical to those in the adjacent slightly atrophied epidermis. (X2000.) 



Bui,!,. U. S. B. F., 1913. 



PtATE XX. 




14 



Buix. U. S. B. F., 191; 



Plate XXI. 





23 





27 



35 








<^ 




30 



^ 



34 



